2. Aims of Presentation
• Answers to the following questions:-
• (1) Why do we test antimicrobial
susceptibility?
• (2) How do we perform antimicrobial
susceptibility tests?
• (3) How can we detect resistance
mechanisms?
• (4) Why & how do we assay antimicrobial
serum levels?
3. Deciding on whether to use an
antibiotic:
• Day 1 Clinical assessment
• Day 2 Positive microbiology
• Day 3 Antibiotic sensitivity tests
• Day 1: guess the disease, guess the bug, guess
the sensitivity pattern
• Day 2: guess the sensitivity pattern
• Day 3: know the full picture: BUT TOO LATE?
4. Antibiotics and the Laboratory
• If there was no antibiotic resistance, there
would be no need for a microbiology lab!
• Infections could be treated “syndromically”
5. Why do we test antimicrobial
susceptibility?
• To direct & predict antimicrobial
chemotherapy.
• To review & monitor epidemiological
trends.
• To set national & local antibiotic policies.
• To test the activity of a new antimicrobial
agent.
• To presumptively identify isolates.
6. But remember…
• Other factors are very important when we
choose an antibiotic
• Will it get to where the infection is?
• Bioavailability
• Cost
• Toxicity
• Likelihood of development of resistance
• Etc…
7. How do we perform antimicrobial
susceptibility tests?
• We can use a number of methods including:-
• Disc susceptibility tests - Kirby-Bauer
- Stokes’
- BSAC.
• Agar Breakpoint method.
• Minimum Inhibitory Concentration (MIC) – Tube
MIC or E-tests.
• Automated methods – Vitek.
• Molecular methods – PCR.
8. Disc Susceptibility Tests
• Agar surface evenly inoculated with the test
organism.
• Antibiotic filter paper discs applied to the plate.
• Plates are incubated & antibiotics diffuse into the
agar.
• Antibiotic concentration decreases at increasing
distance from disc.
• Circular zone of growth appears.
• Size of zone of inhibition indicates susceptibility
of organism.
10. Stokes’ Comparative Method
• Developed in the U.K (1972).
• A variety of media can be used including
Iso-sensitest agar (ISA), ISA & 5% horse
blood & Chocolate ISA.
• Based on dense not confluent growth.
• Use suspension of organism in broth
equivalent in density to an overnight broth
culture.
• Inoculate fastidious organisms direct.
11. Stokes’ Comparative Method
• Use NCTC (National Collection of Type
Cultures) controls e.g. NCTC 6571 Staph
aureus, NCTC 10602 Ps. aeruginosa,
NCTC 10418 E.coli.
• Using a rotary plater apply the control
suspension on the outer edge & the test
suspension in the centre, leaving a gap for
the discs.
14. Stokes’ Comparative Method
• Interpretation based on comparison between
zones seen with the test organism & those of the
known sensitive control.
• Sensitive = zone radius of test, larger, equal, or
not more than 3mm smaller than the control.
• Intermediate = zone radius more than 3mm, but
smaller than the control by more than 3mm.
• Resistant = zone radius of 3mm or less.
15. Disadvantages of Stokes’ Method
• Interpretation not valid for β lactamase-
producing staphylococci (research for
practical) or for tests with polymyxin,
augmentin, teicoplanin or ciprofloxacin.
• No correlation of zone diameter with the
MIC of the organism.
• No standard method for inoculum
preparation- a heavy inoculum decreases
zone of inhibition.
16. Disadvantages of Stokes’ Method
• No standard method for media or
incubation conditions- pre-incubation
decreases the zone of inhibition, pre-
diffusion increases the zone of inhibition.
• Unreliable for detection of resistance to
new antibiotics or newer resistance
mechanisms (ESBL’s).
• No consistent method between labs,
therefore no consistent epidemiology data.
17. BSAC Method
• Developed in the U.K in 1998 by BSAC
working party, through statistical
regression analysis of zone diameter &
MIC data, on hundreds of strains.
• A full up-to-date version of the method is
available at www.bsac.org.uk.
• Use ISA and/or ISA & 5% horse blood &
20mg/l NAD.
• Use standard 0.5 McFarland inoculum
18. BSAC Method
• Use this diluted inoculum to seed the media
(using a rotary plater or by streaking in 3
directions).
• Use standard inoculation & incubation criteria.
• Use standard antibiotic quality controlled discs.
• Use published BSAC tables to interpret zone
sizes.
• http://www.bsac.org.uk/_db/_documents/version
_7_1_february_2008.pdf
20. BSAC Method
• Interpretation is based on semi-confluent growth
of the organism.
• Zones sizes can be measured using a template /
ruler / electronic callipers / automated zone
reader with a scanner & camera (Aura Image,
Oxoid).
• The method is subject to weekly NCTC control
checks for each panel of antibiotics tested.
• These controls are checked against published
values.
21. Rationale of BSAC method
• Based on the
relationship between
the zone diameter of
the disc diffusion test
and the MIC.
• The clinical
breakpoint can be set
as equivalent to a
stated zone diameter
(mm)
22. Advantages of the BSAC Method
• BSAC working party continually review &
update the data.
• Standardised method of reporting.
• Increases the accuracy of epidemiological
data.
• Attempting to standardise protocols and
breakpoints throughout Europe (EUCAST)
– April 2008
23. Minimum Inhibitory Concentration
(MIC)
• The MIC is the lowest concentration of the
antimicrobial required to inhibit growth of the
organism.
• It is used to determine the quantitative activity of
an antimicrobial.
• It is used to confirm resistance or equivocal
results.
• It is used in cases of prolonged treatment or
endocarditis to adjust the dose of therapy.
• It is used to determine the susceptibility of slow-
growing organisms e.g anaerobes
24. Tube MIC
• Set up a series of antibiotic doubling
dilutions in tubes containing liquid media
(ISA or ISA with lysed blood).
• For example 128mg/l, 64, 32, 16, 8, 4, 2,
1, 0.5, 0.25, 0.125, 0.
• Set up tubes for test organism & NCTC
control organism.
25. Tube MIC
• Add standard organism inoculum to each
tube.
• Include an antibiotic free tube i.e organism
only.
• Include an organism free tube i.e antibiotic
only.
• Incubate tubes overnight.
• Examine for presence of growth by
shaking each tube & observing turbidity.
26. Tube MIC
• Check antibiotic free tube has growth.
• Check organism free tube has no growth.
• Check the NCTC control gives the
recommended MIC.
• The MIC is the first tube dilution without
visible growth.
• The tube MIC is very labour intensive,
difficult to get right & prone to error.
27. E-tests (AB Biodisk(Sweden))
• A commercial alternative to tube MIC.
• Consists of a plastic strip 6cm by 0.5cm in
size.
• Exponential gradient of antimicrobial dried
on one side.
• MIC scale printed on the other side.
• The range corresponds to fifteen 2-fold
dilutions.
28. E-tests
• Follow manufacturers’ instructions for
inoculum preparation, media
recommendations & incubation conditions.
• MIC interpretation made where growth of
inhibition ellipses the strip.
• Most E-test require examination with a
hand-lens to look for minute colonies
intersecting the strip.
31. Automated Methods
• Three main methods are in use in the U.K.
• These include the Vitek (Biomerieux), the
Phoenix (Becton-Dickinson) & the
Mastascan Elite (Mast).
• This presentation will focus on the Vitek,
please research the other two methods.
32. Vitek II
• The Vitek I was originally designed by
NASA for use as an on-board space
exploration test system.
• It is based on the use of small thin plastic
cards each containing many wells linked
by capillaries.
• These cards are available as susceptibility
& identification cards.
34. Vitek modules
• The Vitek II consists of:-
• a robotic filling module whereby a standard
suspension of the organism in saline is drawn up
via a vacuum into the card.
• an incubator / reader module containing a
carousel to hold the cards & a photometer to
measure optical density of the sensitivity cards &
the biochemical colour changes of the
identification cards
35. Vitek modules
• A computer module analyses the growth curve &
generates an algorithm devised MIC value. It
also analyses the biochemical id & compares it
to a database.
• An “expert” software analysis module
recognises new / unusual / inconsistent results,
highlights “alert” organisms (e.g. MRSA, VRE,
Gentamicin resistance).
• The “expert” system has built-in antibiotic
interpretation rules.
37. Vitek advantages / disadvantages
• Can give rapid “expert” results for
identification & susceptibility.
• It can be directly linked to the LIMS.
• It decreases the incidence of operator
error & can be operated by an MLA.
• It is very expensive, approx £7 for an id &
sens.
• It needs regular software updates & expert
analysis by a BMS.
38. Why & how do we assay
antimicrobial serum levels?
• We assay to ensure adequate therapeutic levels
& to avoid the accumulation of toxic levels.
• Most antimicrobial agents have a large
therapeutic index & are given in large doses
without causing harm.
• The aminoglycosides, the glycopeptides & some
antifungals have a narrow therapeutic range
which can be close to the toxic range.
39. Dosing interval and steady state plasma concentration
Time
Drugconcentrationinplasma
Therapeutic
range
Therapeutic
failure
Therapeutic
failure
40. Antibiotic Assays
• These agents can damage the 8th cranial
nerve = ototoxicity = deafness.
• They can also damage the kidneys =
nephrotoxicity = renal failure.
• Serum samples are often taken pre-dose
to determine toxicity & post-dose to
determine therapeutic activity.
• We can assay antibiotics in 3 main ways –
bioassay, immunoassay and HPLC
41. Immunoassays
• There are several commercial
immunoassay methods available.
• Most are based on competitive binding of
antibody for antigen (serum) & labelled
antigen (kit).
• The most common commercial method is
the TDX.
43. Calibration curve
• A calibration curve of polarisation versus
concentration is set up within the TDX
using known concentration calibrators.
• Internal controls are run with each assay
to ensure the curve is correct.
• The assay level is calculated by the TDX
via extrapolation from the calibration
curve.
45. References
• British Society for Antimicrobial Chemotherapy
www.bsac.org.uk
• Medical Bacteriology: A Practical Approach Ed P.Hawkey
& D.Lewis, Oxford University Press, 2003.
• Antibiotics & Chemotherapy: Anti-Infective Agents & their
use in therapy R.Finch et al Churchill Livingstone, 2002.
• Textbook of Diagnostic Microbiology C.R Mahon &
G.Manuselis Saunders & Co Ltd 2000.
• Antimicrobial Chemotherapy D. Greenwood Oxford
Medical Publications 1999.
• Clinical Microbiology; E. J. Stokes, G.L. Ridgway &
M.W.D Wren: Arnold; 1993
